Preparation method of neural stem cell

A technology of neural stem cells and plasmic stem cells, which is used in nervous system cells, animal cells, nervous system diseases, etc.

Active Publication Date: 2015-03-18
微能生命科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the process of individual development, there are various forms of stem cells, such as embryonic stem cells, pluripotent stem cells, and committed stem ce...

Method used

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  • Preparation method of neural stem cell
  • Preparation method of neural stem cell
  • Preparation method of neural stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1, the preparation of culture medium

[0087] Preparation of medium A, B, C, D and E:

[0088] The first medium (medium A) consists of animal cell basal medium and solutes. Animal cell basal medium is Knockout DMEM or Knockout DMEM / F12; solutes and their concentrations in animal cell basal medium are as follows: 200ml / L Knockout serum substitute, 10ml / L non-essential amino acids, 0.1mmol / L β-mercaptoethanol , 1mmol / L L-glutamine, 4ng / ml b-FGF.

[0089] The second medium (medium B) consists of animal cell basal medium and solutes. The animal cell basal medium is Neurobasal medium and DMEM / F12 medium with a volume ratio of 1:1; the solute and its concentration in the animal cell basal medium are as follows: 10ml / L N2 supplement, 20ml / L B27 supplement, 1mmol / L L-glutamine, 0.1mmol / Lβ-mercaptoethanol.

[0090] The third medium (medium C) consists of animal cell basal medium and solutes. The animal cell basal medium is Neurobasal medium and DMEM / F12 medium w...

Embodiment 2

[0093]Example 2, Induced Culture of Human Neural Stem Cells

[0094] Step 1. Obtain the third generation of mesenchymal stem cells

[0095] 1. Wash the adipose tissue collected by liposuction with D-Hank’s (or PBS) to remove blood cells and anesthetics, digest with 2g / L type II collagenase at 37°C for 30min, filter through a 100-mesh sieve and collect the filtrate.

[0096] 2. Resuspend the cells obtained in step 1 with D-Hank's solution and wash twice to remove collagenase, centrifuge at 1200rpm at room temperature for 10min, and collect the cells.

[0097] 3. Divide the cells from step 2 in a 2×10 6 The density of each / ml was inoculated in the expansion culture medium (an example of a formula is containing 580ml / L DMEM / F12, 400ml / L MCDB, 20ml / L fetal bovine serum, 1×10 -9 mol / L insulin-transferrin-selenium supplement (ITS), 1×10 -9 mol / L dexamethasone, 1×10 -4 mol / L 2-phosphate ascorbic acid, 20ng / mL interleukin-6, 10ng / mL EGF, 10ng / mL PDGF-BB, 100U / mL penicillin and 100...

Embodiment 3

[0107] Example 3, identification of the terminal differentiation ability of the obtained neural stem cells

[0108] 1. Differentiate into neurons: the cells finally obtained in step 2 of Example 2 were planted in a 6-well plate coated with polyguanine / laminin (PDL / laminin), 5×10 per well 5 Add the fourth medium (ie medium D) to the cells, change the medium every 2 days, and co-culture for 12-16 days, such as 14 days.

[0109] 2. Differentiation to glia: the cells finally obtained in step 2 of Example 2 were planted in a 6-well plate coated with polyguanine / laminin (PDL / laminin), 5×10 per well 5 Add medium E to the cells, change the medium every 2 days, and co-culture for 12-16 days, such as 14 days.

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Abstract

The invention relates to a preparation method of a neural stem cell and particularly discloses a method for inducing a human mesenchymal stem cell to obtain the neutral stem cell. The method comprises the step of sequentially cultivating the mesenchymal stem cell in first, second and third culture media to obtain the neural stem cell. The finally obtained neural stem cell expresses a mark of an early neural stem cell and can be differentiated to functional neuron and neurogliocyte.

Description

technical field [0001] The present invention relates to the field of regenerative medicine. Specifically, it relates to a preparation method of neural stem cells. More specifically, the present invention relates to a method for inducing mesenchymal stem cells into neural stem cells. Background technique [0002] The incidence of degenerative diseases of the nervous system is increasing year by year, mainly manifested as abnormal function of nerve cells or progressive loss of cells, and it is a common clinical refractory disease. For the body itself, dysfunctional nerve cells are difficult to repair by itself, and the limited nerve regeneration ability of the body itself cannot meet the demand for the number of cells needed to repair damage or replace cells. At present, for these refractory diseases, cell nutrition therapy or drug therapy is generally used clinically, but the effect is minimal, and there is no safe and effective treatment method yet. Therefore, the treatme...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/0793C12N5/079A61K35/30A61P25/00A61P25/16A61P25/28
Inventor 赵春华
Owner 微能生命科技集团有限公司
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